Anthranilic acid amides and their use as VEGF receptor tyrosine kinase inhibitors

ABSTRACT

The invention relates to anthranilic acid amide derivatives of formula I, 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  represents H or lower alkyl, R 2  represents H or lower alkyl, R 3  represents perfluoro lower alkyl, and X is O or S, or an N-oxide or a tautomer thereof, to salts of such anthranilic acid amides, their N-oxides and their tautomers; 
             processes for the preparation thereof, the application thereof for the treatment of the human or animal body, the use thereof—alone or in combination with one or more other pharmaceutically active compounds—for the treatment especially of a neoplastic disease, such as a tumor disease, of retinopathy or age-related macular degeneration; a method for the treatment of such a disease in animals, especially in humans, and the use of such a compound—alone or in combination with one or more other pharmaceutically active compounds—for the manufacture of a pharmaceutical preparation for the treatment of a neoplastic disease, of retinopathy or age-related macular degeneration.

This application is a continuation of U.S. application No. 10/494,591,filed May 5, 2004, which is a 371 of International Application No.PCT/EP02/12444, filed Nov. 7, 2002.

The invention relates to new anthranilic acid amide derivatives,processes for the preparation thereof, the application thereof in aprocess for the treatment of the human or animal body, the usethereof—alone or in combination with one or more other pharmaceuticallyactive compounds—for the treatment especially of a neoplastic disease,such as a tumor disease, of retinopathy and age-related maculardegeneration; a method for the treatment of such a disease in animals,especially in humans, and the use of such a compound—alone or incombination with one or more other pharmaceutically active compounds—forthe manufacture of a pharmaceutical preparation (medicament) for thetreatment of a neoplastic disease, of retinopathy or age-related maculardegeneration.

Certain diseases are known to be associated with deregulatedangiogenesis, for example diseases-caused by ocular neovascularisation,such as retinopathies (including diabetic retinopathy), age-relatedmacular degeneration, psoriasis, haemangioblastoma, haemangioma,arteriosclerosis, inflammatory diseases, such as rheumatoid or rheumaticinflammatory diseases, especially arthritis, such as rheumatoidarthritis, or other chronic inflammatory disorders, such as chronicasthma, arterial or post-transplantational atherosclerosis,endometriosis, and especially neoplastic diseases, for example so-calledsolid tumours and liquid tumours (such as leucemias).

At the centre of the network regulating the growth and differentiationof the vascular system and its components during embryonic development,normal growth and in a wide number of pathological anomalies anddiseases, lies the angiogenic factor known as “Vascular EndothelialGrowth Factor” (VGEF), a dimeric, disulfide-linked 46-kDa glycoprotein,along with its cellular receptors (see Breier, G., et al., Trends inCell Biology 6, 454-6 [1996]).

VEGF receptors are transmembranous receptor tyrosine kinases. Varioustypes of VEGF receptor are known, e.g. VEGFR-1, VEGFR-2, and VEGFR-3.

A large number of human tumors, especially gliomas and carcinomas,express high levels of VEGF and its receptors. This has led to thehypothesis that the VEGF released by tumor cells could stimulate thegrowth of blood capillaries and the proliferation of tumor endotheliumin a paracrine manner and thus; through the improved blood supply,accelerate tumor growth. Direct evidence of the role of VEGF as a tumorangiogenesis factor in vivo has been obtained from studies in which VEGFactivity was inhibited by antibodies.

Angiogenesis is regarded as an absolute prerequisite for those tumorswhich grow beyond a maximum diameter of about 1-2 mm; up to this limit,oxygen and nutrients may be supplied to the tumor cells by diffusion.

Three principal mechanisms play an important part in the activity ofangiogenesis inhibitors against tumors: 1) inhibition of the growth ofvessels, especially capillaries, into a vascular resting tumors, withthe result that there is no net tumor growth owing to the balance thatis achieved between apoptosis and proliferation; 2) prevention of themigration of tumor cells owing to the absence of blood flow to and fromtumors; and 3) inhibition of endothelial cell proliferation, thusavoiding the paracrine growth-stimulating effect exerted on thesurrounding tissue by the endothelial cells which normally line thevessels.

In WO00/27820 compounds are described belonging to the class ofanthranilic acid amides which compounds are reported to inhibit theactivity of the VEGF receptor tyrosine kinase, the growth of tumors andVEGF-dependent cell proliferation.

Surprisingly, it has now been found that the anthranilic acid amidederivatives of formula I, described below, have advantageouspharmacological properties and inhibit, for example, the activity of theVEGF receptor tyrosine kinase, the growth of tumors and VEGF-dependentcell proliferation.

The anthranilic acid amide derivatives of formula I are suitable, forexample, to be used in the treatment of diseases, especially fordiseases in the treatment and also for the prevention of which, aninhibition of angiogenesis and/or of the VEGF receptor tyrosine kinaseshows beneficial effects.

The invention pertains to anthranilic acid amides of formula I,

wherein

-   -   R₁ represents H or lower alkyl,    -   R₂ represents H or lower alkyl,    -   R₃ represents perfluoro lower alkyl, and    -   X is O or S,    -   to N-oxides and tautomers thereof,    -   and to salts of such anthranilic acid amides, their N-oxides and        their tautomers.

The general terms used hereinbefore and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise indicated:

The prefix “lower” denotes a radical having up to and including amaximum of 7, especially up to and including a maximum of 4 carbonatoms, the radicals in question being either linear or branched withsingle or multiple branching.

Where the plural form is used for compounds, salts, and the like, thisis taken to mean also a single compound, salt, or the like.

Any asymmetric carbon atoms (for example in compounds of formula I,wherein R₉ is lower alkyl) may be present in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration. Thecompounds may thus be present as mixtures of isomers or as pure isomers,preferably as enantiomer-pure diastereomers.

The invention relates also to possible tautomers of the compounds offormula I. The term “tautomers” as used herein relates in particular tocompounds of formula I wherein R₁ represents H and X is O or S, whichcompounds does also exist to some extend, if not totally, in thetautomeric form shown below (I—Tautomer), wherein the further radicalsand symbols have the meaning as defined herein.

In the preferred embodiment, alkyl has up to a maximum of 12 carbonatoms, and is especially lower alkyl.

Lower alkyl is preferably alkyl with from and including 1 up to andincluding 7, preferably from and including 1 to and including 4, and islinear or branched; preferably, lower alkyl is butyl, such as n-butyl,sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl,ethyl or preferably methyl.

The term “perfluoro lower alkyl” as used herein means a lower alkylradical wherein all hydrogen atoms are replaced by fluoro atoms.

Halogen is especially fluorine, chlorine, bromine, or iodine, especiallyfluorine, chlorine, or bromine.

Such salts are formed, for example, as acid addition salts, preferablywith organic or inorganic acids, from compounds of formula I with abasic nitrogen atom, especially the pharmaceutically acceptable salts.Suitable inorganic acids are, for example, halogen acids, such ashydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organicacids are, for example, carboxylic, phosphonic, sulfonic or sulfamicacids, for example acetic acid, propionic acid, octanoic acid, decanoicacid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid,succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid,malic acid, tartaric acid, citric acid, amino acids, such as glutamicacid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleicacid, cyclohexanecarboxylic, acid, adamantanecarboxylic acid, benzoicacid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylaceticacid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,1,5-naphthalenedisulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid,methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid,N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamicacid, or other organic protonic acids, such as ascorbic acid.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred.

In view of the close relationship between the novel compounds in freeform and those in the form of their salts, including those salts thatcan be used as intermediates, for example in the purification oridentification of the novel compounds, any reference to the freecompounds hereinbefore and hereinafter is to be understood as referringalso to the corresponding salts, as appropriate and expedient.

The compounds of formula I and N-oxides thereof have valuablepharmacological properties, as described hereinbefore and hereinafter.

The efficacy of the compounds of the invention as inhibitors ofVEGF-receptor tyrosine kinase activity can be demonstrated as follows:

Test for activity against VEGF-receptor tyrosine kinase. The test isconducted using Flt-1 VEGF-receptor tyrosine kinase. The detailedprocedure is as follows: 30 μl kinase solution (10 ng of the kinasedomain of Flt-1, Shibuya et al., Oncogene 5, 519-24 [1990]) in 20 mMTris·HCl pH 7.5, 3 mM manganese dichloride (MnCl₂), 3 mM magnesiumchloride (MgCl₂), 10 μM sodium vanadate, 0.25 mg/ml polyethylenglycol(PEG) 20000, 1 mM dithiothreitol and 3 μg/μl poly(Glu,Tyr) 4:1 (Sigma,Buchs, Switzerland), 8 μM [³³P]-ATP (0.2 μCi), 1% dimethyl sulfoxide,and 0 to 100 μM of the compound to be tested are incubated together for10 minutes at room temperature. The reaction is then terminated by theaddition of 10 μl 0.25 M ethylenediaminetetraacetate (EDTA) pH 7. Usinga multichannel dispenser (LAB SYSTEMS, USA), an aliquot of 20 μl isapplied to a PVDF (=polyvinyl difluoride) Immobilon P membrane(Millipore, USA), through a Millipore microtiter filter manifold andconnected to a vacuum. Following complete elimination of the liquid, themembrane is washed 4 times successively in a bath containing 0.5%phosphoric acid (H₃PO₄) and once with ethanol, incubated for 10 minuteseach time while shaking, then mounted in a Hewlett Packard TopCountManifold and the radioactivity measured after the addition of 10 μlMicroscint® (β-scintillation counter liquid). IC₅₀-values are determinedby linear regression analysis of the percentages for the inhibition ofeach compound in three concentrations (as a rule 0.01, 0.1, and 1 μmol).The IC₅₀-values that can be found with compounds of formula I are in therange of 0.001 to 1 μM, preferably in the range from 0.001 to 0.1 μM.

The antitumor efficacy of the compounds of the invention can bedemonstrated in vivo as follows:

In vivo activity in the nude mouse xenotransplant model: female BALB/cnude mice (8-12 weeks old), Novartis Animal Farm, Sisseln, Switzerland)are kept under sterile conditions with water and feed ad libitum. Tumorsare induced either by subcutaneous injection of tumor cells into mice(for example, Du 145 prostate carcinoma cell line (ATCC No. HTB 81; seeCancer Research 37, 4049-58 (1978)) or by implanting tumor fragments(about 25 mg) subcutaneously into the left flank of mice using a13-gauge trocar needle under Forene® anaesthesia (Abbott, Switzerland).Treatment with the test compound is started as soon as the tumor hasreached a mean volume of 100 mm³. Tumor growth is measured two to threetimes a week and 24 hours after the last treatment by determining thelength of two perpendicular axes. The tumor volumes are calculated inaccordance with published methods (see Evans et al., Brit. J. Cancer 45466-8 [1982]). The antitumor efficacy is determined as the mean increasein tumor volume of the treated animals divided by the mean increase intumor volume of the untreated animals (controls) and, aftermultiplication by 100, is expressed as T/C %. Tumor regression (given in%) is reported as the smallest mean tumor volume in relation to the meantumor volume at the start of treatment. The test compound isadministered daily by gavage.

As an alternative other cell lines may also be used in the same manner,for example:

-   -   the MCF-7 breast adenocarcinoma cell line (ATCC No. HTB 22; see        also J. Natl. Cancer Inst. (Bethesda) 51, 1409-16 [1973]);    -   the MDA-MB 468 breast adenocarcinoma cell line (ATCC No. HTB        132; see also In Vitro 14, 911-15 [1978]);    -   the MDA-MB 231 breast adenocarcinorna cell line (ATCC No. HTB        26; see also J. Natl. Cancer Inst. (Bethesda) 53, 661-74        [1974]);    -   the Colo 205 colon carcinoma cell line (ATCC No. CCL 222; see        also Cancer Res. 38, 1345-55 [1978]);    -   the HCT 116 colon carcinoma cell line (ATCC No. CCL 247; see        also Cancer Res. 41, 1751-6 [1981]);    -   the DU145 prostate carcinoma cell line DU 145 (ATCC No. HTB 81;        see also Cancer Res. 37, 4049-58 [1978]); and    -   the PC-3 prostate carcinoma cell line PC-3 (ATCC No. CRL 1435;        see also Cancer Res. 40, 524-34 [1980]).

The inhibition of VEGF-induced KDR-receptor autophosphorylation can beconfirmed with a further in vitro experiment in cells: transfected CHOcells, which permanently express human VEGF receptor (KDR), are seededin complete culture medium (with 10% fetal calf serum=FCS) in 6-wellcell-culture plates and incubated at 37° C. under 5% CO₂ until they showabout 80% confluency. The compounds to be tested are then diluted inculture medium (without FCS, with 0.1% bovine serum albumin) and addedto the cells. (Controls comprise medium without test compounds). Aftertwo hours' incubation at 37° C., recombinant VEGF is added; the finalVEGF concentration is 20 ng/ml). After a further five minutes'incubation at 37° C., the cells are washed twice with ice-cold PBS(phosphate-buffered saline) and immediately lysed in 100 μl lysis bufferper well. The lysates are then centrifuged to remove the cell nuclei,and the protein concentrations of the supernatants are determined usinga commercial protein assay (BIORAD). The lysates can then either beimmediately used or, if necessary, stored at −20° C.

A sandwich ELISA is carried out to measure the KDR-receptorphosphorylation: a monoclonal antibody to KDR (for example Mab1495.12.14; prepared by H. Towbin) is immobilized on black ELISA plates(OptiPlate™ HTRF-96 from Packard). The plates are then washed and theremaining free protein-binding sites are saturated with 1% BSA in PBS.The cell lysates (20 μg protein per well) are then incubated in theseplates overnight at 4° C. together with an antiphosphotyrosineantibody-coupled with alkaline phosphatase (PY20:AP from TransductionLaboratories). The (plates are washed again and the) binding of theantiphosphotyrosine antibody to the captured phosphorylated receptor isthen demonstrated using a luminescent AP substrate (CDP-Star, ready touse, with Emerald II; TROPIX). The luminescence is measured in a PackardTop Count Microplate Scintillation Counter (Top Count). The differencebetween the signal of the positive control (stimulated with VEGF) andthat of the negative control (not stimulated with VEGF) corresponds toVEGF-induced KDR-receptor phosphorylation (=100%). The activity of thetested substances is calculated as % inhibition of VEGF-inducedKDR-receptor phosphorylation, wherein the concentration of substancethat induces half the maximum inhibition is defined as the ED50(effective dose for 50% inhibition).

A compound of formula I or a N-oxide thereof inhibits to varying degreesalso other tyrosine kinases involved in signal transduction which aremediated by trophic factors, for example, kinases from the Src family,especially c-Src kinase, Lck, and Fyn; also kinases of the EGF family,for example, c-erbB2 kinase (HER-2), c-erbB3 kinase, c-erbB4 kinase;insulin-like growth factor receptor kinase (IGF-1 kinase), especiallymembers of the PDGF-receptor tyrosine kinase family, such asPDGF-receptor kinase, CSF-1-receptor kinase, Kit-receptor kinase andVEGF-receptor kinase; and also serine/threonine kinases, all of whichplay a role in growth regulation and transformation in mammalian cells,including human cells.

On the basis of these studies, a compound of formula I according to theinvention shows therapeutic efficacy especially against disordersdependent on protein kinase, especially proliferative diseases.

The usefulness of a compound of the formula I in the treatment ofarthritis as an example of an inflammatory rheumatic or rheumatoiddisease can be demonstrated as follows:

The well-known rat adjuvant arthritis model (Pearson, Proc. Soc. Exp.Biol. 91, 95-101 (1956)) is used to test the anti-arthritic activity ofcompounds of the formula I, or salts thereof. Adjuvant Arthritis can betreated using two different dosing schedules: either (i) starting timeof immunisation with adjuvant (prophylactic dosing); or from day 15 whenthe arthritic response is already established (therapeutic dosing).Preferably a therapeutic dosing schedule is used. For comparison, acyclooxygenase-2 inhibitor, such as5-bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)phenyl]thiophene ordiclofenac, is administered in a separate group.

In detail, male Wistar rats (5 animals per group, weighing epproximately200 g, supplied by Iffa Credo, France) are injected i.d.(intra-dermally) at the base of the tail with 0.1 ml of mineral oilcontaining 0.6 mg of lyophilised heat-killed Mycobacterium tuberculosis.The rats are treated with the test compound (3, 10 or 30 mg/kg p.o. onceper day), or vehicle (water) from day 15 to day 22 (therapeutic dosingschedule). At the end of the experiment, the swelling of the tarsaljoints is measured by means of a mico-calliper. Percentage inhibition ofpaw swelling is calculated by reference to vehicle treated arthriticanimals (0% inhibition) and vehicle treated normal animals (100%inhibition).

On the basis of these studies, a compound of formula I surprisingly isappropriate for the treatment of inflammatory (especially rheumatic orrheumatoid) diseases.

On the basis of their efficacy as inhibitors of VEGF-receptor tyrosinekinase activity the compounds of the formula I primarily inhibit thegrowth of blood vessels and are thus, for example, effective against anumber of diseases associated with deregulated angiogenesis, especiallydiseases caused by ocular neovascularisation, especially retinopathies,such as diabetic retinopathy or age-related macular degeneration,psoriasis, haemangioblastoma, such as haemangioma, mesangial cellproliferative disorders, such as chronic or acute renal diseases, e.g.diabetic nephropathy, malignant nephrosclerosis, thromboticmicroangio-pathy syndromes or transplant rejection, or especiallyinflammatory renal disease, such as glomerulonephritis, especiallymesangioproliferative glomerulonephritis, haemolytic-uraemic syndrome,diabetic nephropathy, hypertensive nephrosclerosis, atheroma, arterialrestenosis, autoimmune diseases, acute inflammation, fibrotic disorders(e.g. hepatic cirrhosis), diabetes, endometriosis, chronic asthma,arterial or post-transplantational atherosclerosis, neurodegenerativedisorders and especially neoplastic diseases like leukaemias, especiallyacute lymphoblastic leukaemia, acute myeloid leukaemia and chronicmyeloid leukaemia, and other “liquid tumours”, especially thoseexpressing c-kit, KDR or fit-1, and solid tumours, especially breastcancer, cancer of the colon, lung cancer (especially small-cell lungcancer), cancer of the prostate or Kaposi's sarcoma. A compound offormula I (or an N-oxide thereof) inhibits the growth of tumours and isespecially suited to preventing the metastatic spread of tumours and thegrowth of micrometastases.

A compound of formula I can be administered alone or in combination withone or more other therapeutic agents, possible combination therapytaking the form of fixed combinations or the administration of acompound of the invention and one or more other therapeutic agents beingstaggered or given independently of one another, or the combinedadministration of fixed combinations and one or more other therapeuticagents. A compound of formula I can besides or in addition beadministered especially for tumor therapy in combination withchemotherapy, radiotherapy, immunotherapy, surgical intervention, or acombination of these. Long-term therapy is equally possible as isadjuvant therapy in the context of other treatment strategies, asdescribed above. Other possible treatments are therapy to maintain thepatient's status after tumor regression, or even chemopreventivetherapy, for example in patients at risk.

Therapeutic agents for possible combination are especially one or morecytostatic or cytotoxic compounds, for example, a chemotherapeutic agentor several selected from the group which includes, but is not limitedto, an inhibitor of polyamine biosynthesis, an inhibitor of a proteinkinase, especially of serine/threonine protein kinase, such as proteinkinase C, or of tyrosine protein kinase, such as the epidermal growthfactor receptor tyrosine kinase, a cytokine, a negative growthregulator, such as TGF-β or IFN-β, an aromatase inhibitor, an inhibitorof the interaction of an SH2 domain with a phosphorylated protein,antiestrogens, topoisomerase I inhibitors, topoisomerase II inhibitors,microtubule active agents, alkylating agents, antineoplasticantimetabolites, platin compounds, other anti-angiogenic compounds,gonadorelin agonists, anti-androgens, bisphosphonates. and trastuzumab.

With the groups of preferred compounds of formula I and N-oxides thereofmentioned hereinafter, definitions of substituents from the generaldefinitions mentioned hereinbefore may reasonably be used, for example,to replace more general definitions with more specific definitions orespecially with definitions characterized as being preferred;

Furthermore, the invention relates to the use of a compound of formulaI, wherein the radicals and symbols have the meanings as defined above,or a N-oxide or a pharmaceutically acceptable salt thereof for thepreparation of a pharmaceutical product for the treatment of retinopathyor age-related macula degeneration.

Furthermore, the invention relates to a method for the treatment of aneoplastic disease which responds to an inhibition of the VEGF-receptortyrosine kinase activity, which comprises administering a compound offormula I or a N-oxide or a pharmaceutically acceptable salt thereof,wherein the radicals and symbols have the meanings as defined above, ina quantity effective against the said disease, to a warm-blooded animalrequiring such treatment.

Furthermore, the invention relates to a method for the treatment ofretinopathy or age-related macular degeneration, which comprisesadministering a compound of formula I or a N-oxide or a pharmaceuticallyacceptable salt thereof, wherein the radicals and symbols have themeanings as defined above, in a quantity effective against saiddiseases, to a warm-blooded animal requiring such treatment.

The invention relates in particular to a compound of formula I, wherein

-   -   R₁ represents H or lower alkyl,    -   R₂ represents H or lower alkyl,    -   R₃ represents trifluoromethyl, and    -   X is O,    -   to an N-oxide or a tautomer thereof,    -   and to a salt of such compound, its N-oxide or its tautomer.

Preferably, the invention relates in particular to a compound of formulaI, wherein

-   -   R₁ represents H or methyl,    -   R₂ represents H or methyl,    -   R₃ represents trifluoromethyl, and    -   X is O,    -   to a tautomer thereof,    -   and to a salt of such compound or its tautomer.

More specifically, preference is given to the following compounds offormula I:

-   -   2-[[6-Methoxy-3-pyridinyl]methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide        hydrochloride salt,    -   2-[[6-Methoxy-3-pyridinyl]methyl]amino-N-[2-methyl-3-(trifluoromethyl)phenyl]benzamide,    -   2-[[(1,6-Dihydro-6-oxo-3-pyridinyl)methyl]amino]-N-[3-(trifluoromethyl)phenyl]benzamide,        and    -   2-[[(1,6-Dihydro-6-oxo-3-pyridinyl)methyl]amino]-N-[2-methyl-3-(trifluoromethyl)phenyl]-benzamide,    -   and the tautomers thereof,    -   or a salt of such compound or its tautomer.

A compound of the invention may be prepared by processes that, thoughnot applied hitherto for the new compounds of the present invention, areknown per se, especially a process characterized in that for thesynthesis of a compound of the formula I wherein R₁ represents loweralkyl and the remaining symbols R₂ and R₃ are as defined for a compoundof the formula I, a compound of the formula II

-   -   wherein R₂ and R₃ are as defined for a compound of the formula        I, is reacted with a carbonyl compound of the formula III

-   -   wherein X represents O or S and R₁ is lower alkyl in the        presence of a reducing agent,    -   wherein the starting compounds of formula II and III may also be        present with functional groups in protected form, if necessary,        and/or in the form of salts, provided a salt-forming group is        present and the reaction in salt form is possible;    -   wherein any protecting groups in a protected derivative of a        compound of the formula I are removed;    -   and, if so desired, an obtainable compound of formula I is        converted into another compound of formula I or a N-oxide        thereof, a free compound of formula I is converted into a salt,        an obtainable salt of a compound of formula I is converted into        the free compound or another salt, and/or a mixture of isomeric        compounds of formula I is separated into the individual isomers.

DETAILED DESCRIPTION OF THE REDUCTIVE ALKYLATION

In the more detailed description of the process below, R₁, R₂, R₃ and Xare as defined for compounds of formula I, unless otherwise indicated.

The carbonyl compound of the formula III may also be present in the formof reactive derivative; however, the free aldehyde or ketone ispreferred.

Reactive derivatives of the compounds of formula III are, for example,corresponding bisulfite adducts or especially semiacetals, acetals,semiketals or ketals of compounds of formula III with alcohols, forexample lower alkanols; or thioacetals or thioketals of compounds offormula III with mercaptans, for example lower alkanesulfides.

The reductive alkylation is preferably carried out with hydrogenation inthe presence of a catalyst, especially a noble metal catalyst, such asplatinum or especially palladium, which is preferably bonded to acarrier material, such as carbon, or a heavy metal catalyst, such asRaney nickel, at normal pressure or at pressures of from 0.1 to 10MegaPascal (MPa), or with reduction by means of complex hydrides, suchas borohydrides, especially alkali metal cyanoborohydrides, for examplesodium cyanoborohydride, in the presence of a suitable acid, preferablyrelatively weak acids, such as lower alkanecarboxylic acids, especiallyacetic acid, or a sulfonic acid, such as p-toluenesulfonic acid; incustomary solvents, for example alcohols, such as methanol or ethanol,or ethers, for example cyclic ethers, such as tetrahydrofuran, in thepresence or absence of water.

Protecting Groups

If one or more other functional groups, for example carboxy, hydroxy,amino, or mercapto, are or need to be protected in a compound offormulae II or III, because they should not take part in the reactionthese are such groups as are usually used in the sythesis of peptidecompounds, and also of cephalosporins and penicillins, as well asnucleic acid derivatives and sugars.

The protecting groups may already be present in precursors and shouldprotect the functional groups concerned against unwanted secondaryreactions, such as acylations, etherifications, esterifications,oxidations, solvolysis, and similar reactions. It is a characteristic ofprotecting groups that they lend themselves readily, i.e. withoutundesired secondary reactions, to removal, typically by solvolysis,reduction, photolysis or also by enzyme activity, for example underconditions analogous to physiological conditions, and that they are notpresent in the end-products.The specialist knows, or can easilyestablish, which protecting groups are suitable with the reactionsmentioned hereinabove and hereinafter.

The protection of such functional groups by such protecting groups, theprotecting groups themselves, and their removal reactions are describedfor example in standard reference works, such as J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, London and NewYork 1973, in T. W. Greene, “Protective Groups in Organic Synthesis”,Wiley, New York 1981, in “The Peptides”; Volume 3 (editors: E. Gross andJ. Meienhofer), Academic Press, London and New York 1981, in “Methodender organischen Chemie” (Methods of organic chemistry), Houben Weyl, 4thedition, Volume 15/I, Georg Thieme Verlag, Stuttgart 1974, in H.-D.Jakubke and H. Jescheit, “Aminosäuren, Peptide, Proteine” (Amino acids,peptides, proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel1982, and in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharideund Derivate” (Chemistry of carbohydrates: monosaccharides andderivatives), Georg Thieme Verlag, Stuttgart 1974.

Additional Process Steps

Salts of a compound of formula I with a salt-forming group may beprepared in a manner known per se. Acid addition salts of compounds offormula I may thus be obtained by treatment with an acid or with asuitable anion exchange reagent. A salt with two acid molecules (forexample a dihalogenide of a compound of formula I) may also be convertedinto a salt with one acid molecule per compound (for example amonohalogenide); this may be done by heating to a melt, or for exampleby heating as a solid under a high vacuum at elevated temperature, forexample from 130 to 170° C., one molecule of the acid being expelled permolecule of a compound of formula I.

Salts can usually be converted to free compounds, e.g. by treating withsuitable basic agents, for example with alkali metal carbonates, alkalimetal hydrogencarbonates, or alkali metal hydroxides, typicallypotassium carbonate or sodium hydroxide.

An anthranilic acid amide of formula I wherein R₁ represents lower alkyland the remaining symbols R₂ and R₃ are as defined for a compound of theformula I, obtained by reaction of the compounds of formula II andformula III, can be further reacted in accordance with the followingprocess providing an anthranilic acid amide of formula I wherein R₁represents H. The anthranilic acid amide of formula I wherein R₁represents lower alkyl is treated with trimethylsilyl iodide for about20 to 35 hours at a temperature between 45° C. and 70° C. in a suitablesolvent, e.g. a halogenated alkane, like chloroform, optionally followedby treatment with methanol.

General Process Conditions

All process steps described here can be carried out under known reactionconditions, preferably under those specifically mentioned, in theabsence of or usually in the presence of solvents or diluents,preferably such as are inert to the reagents used and able to dissolvethese, in the absence or presence of catalysts, condensing agents orneutralisiing agents, for example ion exchangers, typically cationexchangers, for example in the H⁺ form, depending on the type ofreaction and/or reactants at reduced, normal, or elevated temperature,for example in the range from −100° C. to about 190° C., preferably fromabout −80° C. to about 150° C., for example at −80 to −60° C., at roomtemperature, at −20 to 40° C. or at the boiling point of the solventused, under atmospheric pressure or in a closed vessel, whereappropriate under pressure, and/or in an inert atmosphere, for exampleunder argon or nitrogen.

Salts may be present in all starting compounds and transients, if thesecontain salt-forming groups. Salts may also be present during thereaction of such compounds, provided the reaction is not therebydisturbed.

The solvents from which those can be selected which are suitable for thereaction in question include for example water, esters, typically loweralkyl-lower alkanoates, e.g diethyl acetate, ethers, typically aliphaticethers, e.g. diethylether, or cyclic ethers, e.g. tetrahydrofuran,liquid aromatic hydrocarbons, typically benzene or toluene, alcohols,typically methanol, ethanol or 1- or 2-propanol, nitriles, typicallyacetonitrile, halogenated hydrocarbons, typically dichloromethane, acidamides, typically dimethylformamide, bases, typically heterocyclicnitrogen bases, e.g. pyridine, carboxylic acids, typically loweralkanecarboxylic acids, e.g. acetic acid, carboxylic acid anhydrides,typically lower alkane acid anhydrides, e.g. acetic anhydride, cyclic,linear, or branched hydrocarbons, typically cyclohexane, hexane, orisopentane, or mixtures of these solvents, e.g. aqueous solutions,unless otherwise stated in the description of the process. Such solventmixtures may also be used in processing, for example throughchromatography or distribution.

The compounds of formula I, including their salts, are also obtainablein the form of hydrates, or their crystals can include for example thesolvent used for crystallization (present as solvates).

In the preferred embodiment, a compound of formula I is preparedaccording to or in analogy to the processes and process steps defined inthe Examples.

The dosage of the active ingredient depends upon a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound employed. A physician, clinician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentration of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

The invention relates also to pharmaceutical compositions comprising aneffective amount, especially an amount effective in the treatment of oneof the above-mentioned disorders, of an anthranilic acid amide offormula I or an N-oxide or a tautomer thereof together withpharmaceutically acceptable carriers that are suitable for topical,enteral, for example oral or rectal, or parenteral administration andthat may be inorganic or organic, solid or liquid.

There are used for oral administration especially tablets or gelatincapsules that comprise the active ingredient together with diluents, forexample lactose, dextrose, mannitol, and/or glycerol, and/or lubricantsand/or polyethylene glycol. Tablets may also comprise binders, forexample magnesium aluminum silicate, starches, such as corn, wheat orrice starch, gelatin, methylcellulose, sodium carboxymethylcelluloseand/or polyvinylpyrrolidone, and, if desired, disintegrators, forexample starches, agar, alginic acid or a salt thereof, such as sodiumalginate, and/or effervescent mixtures, or adsorbents, dyes, flavoringsand sweeteners. It is also possible to use the pharmacologically activecompounds of the present invention in the form of parenterallyadministrable compositions or in the form of infusion solutions. Thepharmaceutical compositions may be sterilized and/or may compriseexcipients, for example preservatives, stabilisers, wetting agentsand/or emulsifiers, solubilisers, salts for regulating the osmoticpressure and/or buffers. The present pharmaceutical compositions, whichmay, if desired, comprise other pharmacologically active substances areprepared in a manner known per se, for example by means of conventionalmixing, granulating, confectioning, dissolving or lyophilisingprocesses, and comprise approximately from 1% to 95%, especially fromapproximately 1% to approximately 20%, active ingredient(s).

Furthermore, the invention relates to a pharmaceutical composition fortreatment of tumours in warm-blooded animals, including humans,comprising an antitumourally effective dose of a compound of the formulaI as described above or a pharmaceutically acceptable salt of such acompound together with a pharmaceutical carrier.

Additionally, the present invention provides an anthranilic acid amideof formula I or an N-oxide or a tautomer thereof, or a pharmaceuticallyacceptable salt of such a compound, for use in a method for thetreatment of the human or animal body.

The present invention also relates to the use of an anthranilic acidamide of formula I or an N-oxide or a tautorer thereof, or apharmaceutically acceptable salt of such a compound, for the preparationof a pharmaceutical product for the treatment of a neoplastic disease,retinopathy or age-related macula degeneration.

In addition to this, the present invention teaches a method for thetreatment of a neoplastic disease which responds to an inhibition of theVEGF-receptor tyrosine kinase activity, which comprises administering ananthranilic acid amide of formula I or a N-oxide or a tautomer thereof,or a pharmaceutically acceptable salt of such anthranilic acid amide,its N-oxide or its tautomer, in a quantity effective against saiddisease, to a warm-blooded animal requiring such treatment.

Starting Materials

New starting materials and/or intermediates, as well as processes forthe preparation thereof, are likewise the subject of this invention. Inthe preferred embodiment, such starting materials are used and reactionconditions so selected as to enable the preferred compounds to beobtained.

Starting materials of the formula III, IV and V are known, commerciallyavailable, or can be synthesized in analogy to or according to methodsthat are known in the art.

For example, a compound of the formula II can be prepared by thereduction of a nitro compound of the formula IV,

wherein R₂ and R₃ have the meanings as given under formula I.

The reduction preferably takes place in the presence of a suitablereducing agent, such as tin(II) chloride or hydrogen in the presence ofan appropriate catalyst, such as Raney nickel (then preferably thehydrogen is used under pressure, e.g. between 2 and 20 bar) or PtO₂, inan appropriate solvent, e.g. an alcohol, such as methanol. The reactiontemperature is preferably between 0 and 80° C., especially 15 to 30° C.

A nitro compound of the formula IV is accessible by reaction of anactived acid derivative of the formula VI,

wherein Y is halogen or another suitable leaving group, is reacted withan amine of the formula V,

wherein R₂ and R₃ are as defined under formula I, e.g. in the presenceof a coupling agent, such as dicyclohexylcarbodiimide, at a temperaturebetween 0° C. and 50° C., preferably at room temperature.

All remaining starting materials of are known, capable of being preparedaccording to known processes, or commercially obtainable; in particular,they can be prepared using processes as described in the Examples.

In the preparation of starting materials, existing functional groupswhich do not participate in the reaction should, if necessary, beprotected. Preferred protecting groups, their introduction and theirremoval are described under “protecting goups” or in the Examples.

All remaining starting materials of are known, capable of being preparedaccording to known processes, or commercially obtainable; in particular,they can be prepared using processes as described in the Examples.

The following Examples serve to illustrate the invention withoutlimiting the invention in its scope.

Temperatures are measured in degrees celsius (° C.). Unless otherwiseindicated, the reactions take place at room temperature.

EXAMPLES Reference Example 12-[[6-Methoxy-3-Pyridinyl]Methyl]Amino-N-[4-Bromo-3-(Trifluoromethyl)Phenyl]Benzamide(Not Claimed)

Sodium cyanoborohydride (8.80 g of 95%, 133 mmol) is added in portionsover 30 minutes to a stirred mixture of acetic acid (3.8 mL),6-methoxy-3-pyridinecarboxaldehyde (Fluka, Buchs, Switzerland; 7.80 g,57 mmol) and 2-amino-N-(4-bromo-3-trifluoromethylphenyl)-benzamide (step1.2; 13.65 g, 38 mmol) in methanol (380 mL) at 25° C. The mixture isstirred for 16 hours. The solvent is evaporated under reduced pressureto give a residue which is treated with a saturated aqueous solution ofsodium hydrogen carbonate (500 mL) and extracted with dichloromethane(3×150 mL). The combined extracts are dried (Na₂SO₄), filtered and thesolvent is evaporated under reduced pressure to yield the crude productthat is purified by column chromatography on silica gel, eluent 5% ethylacetate in dichloromethane and recrystallised from diethylether—hexaneto give the title compound as a beige crystalline solid, m.p. 101-103°C.

Step 1.1: 2-Nitro-N-(4-Bromo-3-Trifluoromethylphenyl)Benzamide

A solution of 3-amino-6-bromobenzotrifluoride (Fluka, Buchs,Switzerland; 24.0 g, 100 mmol) in ethyl acetate (240 mL) is added to astirred aqueous solution of sodium hydroxide (110 mL of 1 M) at roomtemperature. This stirred solution is then treated dropwise over 30minutes with a solution of 2-nitrobenzoyl chloride (Fluka, Buchs,Switzerland; 14.5 mL, 110 mmol) in ethyl acetate (150 mL). The resultingmixture is then stirred for 30 min at ambient temperature. The mixtureis extracted with ethyl acetate (3×100 mL) and the combined extracts aresequentially washed with hydrochloric acid (2×100 mL of 2 M), water(2×100 mL), saturated aqueous sodium hydrogen carbonate solution (2×100mL) and saturated aqueous sodium chloride (1×100 mL), dried (MgSO₄),filtered and the solvent is evaporated under reduced pressure to yieldthe crude product which is purified by recrystallisation from ethylacetate-hexane to give the title compound as a beige crystalline solid,m.p. 157-158° C.

Step 1.2: 2-Amino-N-(4-Bromo-3-Trifluoromethylphenyl)benzamide

A solution of 2-nitro-N-(4-bromo-3-trifluoromethylphenyl)benzamide(intermediate 1a; 32 g, 82 mmol) in methanol (1000 mL) is hydrogenatedat atmospheric pressure over Raney nickel (6 g) at 21° C. The calculatedamount of hydrogen is taken up after 7 hours. The mixture is filteredand the solvent is evaporated under reduced pressure to yield the crudeproduct which is purified by recrystallisation from diethylether-hexaneto give the title compound as a colourless crystalline solid, m.p.142-144° C.

Example 22-[[6-Methoxy-3-Pyridinyl]Methyl]Amino-N-[3-(Trifluoromethyl)Phenyl]benzamideHydrochloride Salt

The title compound is prepared by a method analogous to that describedin Example 1 by utilising the intermediate from step 2.2 and6-methoxy-3-pyridinecarboxaldehyde (Fluka, Buchs, Switzerland); m.p.133-135° C.

Step 2.1: 2-Nitro-N-[3-(Trifluoromethyl)Phenyl]Benzamide

The title compound is prepared analogously to step 1.1 by utilising3-(trifluoromethyl)-benzenamine (Aldrich, Buchs, Switzerland); m.p.134-135° C.

Step 2.2: 2-Amino-N-[(3-trifluoromethyl)Phenyl)Benzamide

The title compound is prepared analogously to step 1.2 by utilising2-nitro-N-[(3-trifluoromethyl)phenyl)benzamide (step 2.1); m.p. 132-133°C.

Example 32-[[6-Methoxy-3-Pyridinyl]Methyl]Amino-N-[2-Methyl-3-(Trifluoromethyl)Phenyl]-Benzamide

The title compound is prepared by a method analogous to that describedin Example 1 by utilising the intermediate from step 3.2 and6-methoxy-3-pyridinecarboxaldehyde (Aldrich, Buchs, Switzerland); m.p.134-135° C.

Step 3.1: 2-Nitro-N-[2-Methyl-3-(Trifluoromethyl)Phenyl]Benzamide

The title compound is prepared analogously to step 1.1 by utilising2-methyl-3-(trifluoromethyl)benzenamine (Fluorochem, Derbyshire,England); m.p. 188-189° C.

Step 3.2: 2-Amino-N-[2-Methyl-(3-Trifluoromethyl)Phenyl)Benzamide

The title compound is prepared analogously to step 1.2 by utilising2-nitro-N[2-methyl-3-(trifluoromethyl)phenyl]benzamide (step 2.1); m.p.128-129° C.

Reference Example 42-[[(1,6-Dihydro-6-Oxo-3-Pyridinyl)Methyl]amino]-N-4-Propynyl-3-(Trifluoromethyl)Phenyl]Benzamide(Not Claimed)

A mixture of2-[[6-methoxy-3-pyridinyl]methyl]amino-N[4-(1-propynyl)-3-(trifluoromethyl)-phenyl]benzamide(step 4.1; 1.10 g,2.5 mmol) and trimethylsilyl iodide (Fluka, Buchs,Switzerland; 1.0 mL, 7.5 mmol) in chloroform (30 mL) is stirred at 60°C. for 16 hours under an argon atmosphere. The cooled mixture is thentreated with methanol (2 mL) and stirred at room temperature for 10minutes. The solvent is evaporated under reduced pressure and theresidue is treated with an aqueous solution of ammonia (100 mL of 5%)and extracted with ethyl acetate (3×100 mL). The combined extracts arewashed with saturated aqueous sodium chloride (50 mL), dried (MgSO₄),filtered and the solvent is evaporated under reduced pressure to yieldthe crude product which is purified by column chromatography on silicagel, eluent ethyl acetate and recrystallised from hot ethylacetate-hexane to give the title compound as a colourless crystallinesolid; m.p. 208-212° C.

Step 4.1:2-[[6-Methoxy-3-Pyridinyl]Methyl]Amino-N-[4-(1-Propynyl)-3-(Trifluoromethyl)-Phenyl]Benzamide

A stirred solution of2-[[6-methoxy-3-pyridinyl]methyl]amino-N-4-bromo-3-(trifluoromethyl)-phenyl]benzamide(Reference Example 1; 3.98 g, 8.3 mmol) in dry toluene (200 mL) ispurged with argon for 20 minutes at 25° C. Tributyl-1-propynylstannane(4.1 g of 80%, 9.96 mmol) and tetrakis(triphenylphosphine)palladium (0)(260 mg) are then added and the resulting mixture is heated at 100° C.for 17 hours under an argon atmosphere. The mixture is then cooled,treated with an aqueous solution of sodium hydroxide (85 mL of 0.1 M)and purged with air for 2 hours. The resulting mixture is extracted withethyl acetate (3×100 mL). The organic phase is sequentially washed withwater (2×40 mL) and saturated aqueous sodium chloride (1×40 mL), dried(Na₂SO₄), filtered and the solvent is evaporated under reduced pressureto yield the crude product which is purified by column chromatography onsilica gel, eluent 33% ethyl acetate in hexane and recrystallised fromdiethylether-hexane to give the title compound as a pale-yellowcrystalline solid; m.p. 123-124° C.

Example 52-[[(1.6-Dihydro-6-Oxo-3-Pyridinyl)Methyl]Amino]-N-[3-(Trifluoromethyl)Phenyl]-Benzamide

The title compound is prepared by a method analogous to that describedin Example 4 by utilising2-[[6-methoxy-3-pyridinyl]methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide(Example 2); m.p. 171-172° C.

Example 62-[[(1,6-Dihydro-6-Oxo-3-Pyridinyl)Methyl]Amino]-N-[2-Methyl-3-(Trifluoromethyl)-Phenyl]Benzamide

The title compound is prepared by a method analogous to that describedin Example 8 by utilising2-[[6-methoxy-3-pyridinyl]methyl]amino-N-[2-methyl-3-(trifluoromethyl)phenyl]benzamide(Example 3); m.p. 191-192° C.

Example 7 Soft Capsules

5000 soft gelatin capsules, each comprising as active ingredient 0.05 gof one of the compounds of formula I mentioned in the precedingExamples, are prepared as follows:

Composition Active ingredient 250 g Lauroglycol 2 liters

Preparation process: The pulverized active ingredient is suspended inLauroglykol® (propylene glycol laurate, Gattefossé S. A., Saint Priest,France) and ground in a wet pulverizer to produce a particle size ofabout 1 to 3 μm. 0.419 g portions of the mixture are then introducedinto soft gelatin capsules using a capsule-filling machine.

1. A method for the treatment of retinopathy or age-related maculadegeneration comprising administering an anthranilic acid amide offormula I,

wherein R₁ represents H or lower alkyl, R₂ represents H or lower alkyl,R₃ represents perfluoro lower alkyl, and X is O or S, or an N-oxide or atautomer thereof, or a salt of such anthranilic acid amide, its N-oxideor its tautomer, or a pharmaceutically acceptable salt of such acompound.
 2. A method for the treatment of a neoplastic disease whichresponds to an inhibition of the VEGF-receptor tyrosine kinase activity,which comprises administering an anthranilic acid amide of formula I,

wherein R₁ represents H or lower alkyl, R₂ represents H or lower alkyl,R₃ represents perfluoro lower alkyl, and X is O or S, or an N-oxide or atautomer thereof, or a salt of such anthranilic acid amide, its N-oxideor its tautomer, in a quantity effective against the said disease, to awarm-blooded animal requiring such treatment wherein said neoplasticdisease in selected from breast cancer, prostate cancer and coloncancer.